Project Summary – Kim Lentiviruses infect both activated/dividing CD4+ T cells and terminally differentiated/nondividing myeloid cells during the course of their pathogenesis. As we previously reported, host SAMHD1 dNTPase restricts viral reverse transcription step specifically in nondividing myeloid cells by depleting cellular dNTPs whereas HIV-2 and some SIVs counteract SAMHD1 by proteosomal degradation through their accessary proteins (e.g. Vpx). In this renewal, we aim to reveal noble mechanistic strategies that lentiviruses employ for their myeloid cell infection and rapid evolution/escape. First, lentiviruses encode an additional polypurine track (PPT) sequence, called central PPT (cPPT), that locates at the center of the viral genome and is used for the additional initiation of the (+) strand DNA synthesis. We previously reported that the concomitant initiation of the (+) strand DNA synthesis from both PPT and cPPT compensates the kinetically delayed HIV-1 reverse transcription in nondividing cells with limited dNTP pools by cutting the size of the (+) strand DNA synthesis from PPT by half. In Aim 1, we will test our hypothesize that the additional cPPT of HIV-1 allows HIV-1 to overcome the SAMHD1-mediated dNTP depletion and complete the (+) strand DNA synthesis even in nondividing myeloid cells without accessary proteins counteracting SAMHD1. Second, we previously reported that the uniquely tight dNTP binding affinity of HIV-1 RT mechanistically contributes to its catalytic capability to execute DNA synthesis even at low dNTP concentrations, which enables HIV-1 to replicate in myeloid cells with very low dNTP pools. Importantly, we also reported that this tight dNTP binding affinity enables HIV-1 RT to efficiently extend mismatch primer post misinsertion, compared to other retroviral RTs, which is responsible for the highly error prone DNA synthesis of HIV-1 RT. Based on these observations, we propose to solve the X-ray structure of HIV-1 RT ternary complex with mismatch primer, which will elucidate the structural nature of the highly error prone HIV-1 replication machinery which is important for viral evolution and escape. Third, while lethal mutagenesis has been observed in other RNA viruses, it remains unclear that the lethal mutagenesis of HIV-1 and lentiviruses can be achieved by pharmacological means. Triphosphate (TP) of Molnupiravir, b-d-N4 hydroxycytidine (NHC) prodrug, is a ribonucleotide substrate and an RNA mutagen for RNA-dependent RNA polymerases of multiple RNA viruses including SARS-CoV-2, which induces viral lethal mutagenesis. Excitingly, our biochemical data demonstrate that host cellular RNA polymerase II also incorporates NHC-TP during RNA synthesis, supporting the likelihood of the NHC-TP incorporation into cellular RNAs by host RNA polymerases. Since lentivirus RNA genomes are synthesized by host DNA-dependent RNA polymerase II, we hypothesize that NHC may be able to induce lethal mutagenesis in lentiviruses. Overal...